Synthesis of PMMA Microparticles with a Narrow Size Distribution by

Article pubs.acs.org/Macromolecules

Synthesis of PMMA Microparticles with a Narrow Size Distribution by Photoinitiated RAFT Dispersion Polymerization with a Macromonomer as the Stabilizer Jianbo Tan,†,‡ Guangyao Zhao,† Yijie Lu,† Zhaohua Zeng,‡ and Mitchell A. Winnik*,† †

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, M5S 3H6, Ontario Canada Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, and Key Laboratory of Designed Synthesis and Application of Polymer Material, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China

‡

S Supporting Information *

ABSTRACT: Macromonomers can serve as efficient and effective stabilizers for dispersion polymerization of monomers such as styrene and methyl methacrylate, but the size distributions of the polymer microparticles obtained tend to be broad. We are interested in functional microbeads which can be used for immunoassays, where the size distribution has to be very narrow. We report a photoinitiated RAFT dispersion polymerization of methyl methacrylate (MMA) in ethanol− water mixtures, with methoxy-poly(ethylene glycol) methacrylate (Mn = 2000 g/mol, EO45) as the reactive steric stabilizer. We identify reaction conditions where one can obtain PMMA microspheres with coefficient of variation in the particle diameter (CVd) less than 3%. Carboxy-functional PMMA microspheres were obtained by a two-stage (seeded) polymerization with methacrylic acid (MAA) added as a comonomer in the second stage. We show that the functional microspheres prepared in this way are effective substrates for the covalent attachment of proteins such as BSA and IgG immunoglobulins. In one set of experiments with a dye-labeled secondary antibody, we found that we could detect 104 IgGs per PMMA microbead.

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INTRODUCTION Monodisperse polymer microspheres have a number of useful and important applications, as separation media, as photonic and ion-exchange support materials, as toners, coatings, and calibration standards, and for multiplex immunoassays.1−7 Micrometer-sized polymer particles can be made by seeded emulsion polymerization as developed by Vanderhoff8 or the activated swelling polymerization method developed by Ugelstad.9 These methods are complex and challenging to implement on a large scale. As an alternative, dispersion polymerization can be used as a one-pot batch process to obtain polymer microspheres with diameters in the range 1−15 μm, often with a very narrow size distribution.10 Dispersion polymerization is defined as a polymerization reaction in which all the reaction ingredients (monomer, initiator, polymeric stabilizer, and solvent) form a homogeneous solution when mixed, but the polymer formed in the reaction, once it exceeds a critical chain length, is not soluble precipitates. What distinguishes dispersion polymerization from precipitation polymerization is the presence of the polymeric stabilizer. This stabilizer is a polymer that is soluble in the reaction medium. During the polymerization reaction, some of the growing polymer chains react with the stabilizer to form a graft copolymer that adsorbs to the precipitating polymer to prevent macroscopic precipitation and provides colloidal © 2014 American Chemical Society

stability for the product. The process of dispersion polymerization can be separated into a nucleation stage and a particle growth stage. In the nucleation stage, coalescence of newly formed particles dominates until the number of colloidally stable particles in the reaction becomes constant. In the growth stage, monomer polymerization occurs in both the particle phase and in solution, and graft copolymer formation continues in solution. All the polymerization products formed in solution following the end of the nucleation stage are swept up by the growing particles. In most dispersion polymerization reactions, the nucleation stage is complete early in the reaction. Since the number of particles remains constant and they all grow at the same rate, the final size distribution can be very narrow. The stabilizers play an important role in this process. They form a solvent-swollen corona surrounding the dense particle core and provide steric or electrosteric stabilization to the particles. The most commonly used stabilizers are homopolymers such as poly(N-vinylpyrrolidone) (PVP),11−13 poly(acrylic acid), or hydroxypropyl cellulose (HPC).14,15 Particle formation in the presence of these stabilizers is normally robust, but because grafting is an inefficient process and because Received: July 11, 2014 Revised: August 21, 2014 Published: September 19, 2014 6856

dx.doi.org/10.1021/ma501432s | Macromolecules 2014, 47, 6856−6866

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homopolymer adsorption can contribute to colloidal stability during the reaction, a large excess of polymer is needed. At the end of the reaction, the particles have to be purified to remove unreacted polymer. We are interested in the synthesis of lanthanide-encoded polymer microbeads to be used in mass cytometry for beadbased assays. These particles have to have very narrow size distributions, narrow and controllable levels of lanthanide ion incorporation, and surface functionality for attachment of bioaffinity agents such as antibodies. We have had good success in synthesizing lanthanide-encoded polystyrene (PS) microspheres by two-stage or three-stage dispersion polymerization in the presence of PVP as the stabilizer.7 Acrylic acid (2 mol %) as a comonomer provided metal-binding sites for the lanthanide ions as well as surface functionality. While a large number of surface carboxyl groups could be detected by titration, they were not very effective for antibody attachment. As a consequence, we have been searching for alternative stabilizers that would carry functionality in the corona of the stabilizer, where they should be more accessible to reactive groups on antibodies and other bioaffinity agents. The literature describes various approaches to employ functional stabilizers for dispersion polymerization, such as block copolymers,16,17 macromolecular chain transfer agents,18,19 and macromonomers.20,21 In this paper, we consider the use of functional macromonomers as stabilizers for the synthesis of polymer microspheres by dispersion polymerization. Macromonomers are polymers with at least one polymerizable group at the end of the polymer. During the reaction, macromonomers copolymerize with monomers and form the graft copolymers that act as the stabilizers for the particles. Since the macromonomers are more reactive and become covalently attached to the particle surface, the amount of macromonomer required for stabilization is much less than that for homopolymer stabilizers. Thus, using macromonomers in the reaction can be more efficient. One can also employ functional macromonomers and in this way introduce functional groups into the corona of the particles. Our intended application requires polymer microparticles with a very narrow size distribution. The lanthanide-encoded PS particles described above were obtained with diameters of ca. 2 μm and coefficients of variation of the particle diameters (CVd = 1−3%). With traditional dispersion polymerization, such as the dispersion polymerization of styrene in ethanol in the presence of excess PVP, one can obtain particles with a CVd as low as 1%. This narrow distribution corresponds to a polydispersity index (PDI = dw/dn) of 1.001. The terms CVd and dw/dn are defined in eqs 1 and 2.

The derivation of eq 3 is shown in Supporting Information. To put our literature review in perspective, we note that PDI = 1.01 corresponds to CVd = 5.8%, PDI = 1.02 corresponds to CVd = 8.2%, and PDI = 1.03 corresponds to CVd = 10%. There have been a number of previous reports of dispersion polymerization using macromonomer stabilizers. These have focused on the increase in efficiency and flexibility in the use of macromonomers in dispersion polymerization. These studies explored how reaction conditions and choice of monomer and macromonomer affected particle size and size uniformity. Particle size distributions were significantly broader than those obtained with traditional polymeric stabilizers, but these studies did not report attempts to optimize reaction conditions for extremely narrow size distribution. While the origin of the broad size polydispersity is not well understood, it may be related to the timing of macromonomer copolymerization with the monomer.22,23 The formation of particles with a narrow size distribution requires that the number of growing particles in the reaction be fixed at a relatively early stage, so that all particles grow in parallel. At the same time, the reaction has to provide enough new graft copolymer throughout the reaction to provide surface coverage as the particles grow larger, but not so much that new particles can be nucleated. Among the various research groups that have used macromonomers as the stabilizer in dispersion polymerization, very few report the synthesis of polymer particles with narrow size distributions. For example, Lacroix-Desmazes et al.24 reported a study of the dispersion polymerization of styrene in ethanol−water mixtures using a series of poly(ethylene oxide) (PEO) maleate ester macromonomers with the general formula: R−MA−EOn−R′, with R = H or n-C12H25, MA = maleic acid, n = 34, 42, and 45, and R′ = H or CH3. They investigated the effect of various reaction parameters, such as the macromonomer concentration and the polarity of the solvent mixture, on the particle size and the particle size distribution. They obtained particles with mean diameters on the order of 2 μm. Their reported particle size distributions ranged from PDI = 1.03, which is relatively narrow to 1.34, which is much broader. Choe’s group25,26 reported the synthesis and characterization of a series of bifunctional vinyl urethane macromonomers with different molecular weights, and applied them to the dispersion polymerization of styrene in ethanol. They obtained polystyrene particles with diameters ranging from 1.4 to 4.4 μm and with PDI values ranging from 1.03 to 2.79. Jung et al.27 also synthesized a bifunctional polyurethane macromonomer and employed it for the dispersion polymerization of methyl methacrylate (MMA) in ethanol. They investigated the effect of macromonomer concentration on particle size and morphology, and found one set of reaction conditions that led to uniform particles. These conditions, with 20 wt % macromonomer based on MMA, led to 5 μm diameter PMMA particles with PDI = 1.01. Liu et al.28 used a methoxypoly(ethylene oxide)40 undecyl-α-methacrylate macromonomer as the stabilizer in dispersion polymerization of styrene in ethanol−water mixtures. They were able to obtain particles of different size and with mean diameters ranging from 100 nm to 1.0 μm using relatively small amounts of macromonomer. These particles, which they called “monodisperse”, had PDI values ranging from 1.05 to 1.14. Richez et al.29 reported the synthesis of PMMA polymer particles by the dispersion polymerization of MMA in dodecane using a poly(dimethylsiloxane) macromonomer (Mw = 5000 g/mol or

n

∑i = 1 (di − dn)2

CVd =

n−1 n

dn =

∑ nidi/n;

/ dn n

(1) n

dw = (∑ nidi 4)/(∑ nidi 3)

i=1

i=1

i=1

(2)

where ni is the number of particles with diameter di. For a Gaussian distribution of particle diameters, CVd is related to dw/dn by the expression dw ≅ 1 + 3(CVd)2 dn

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Table 1. Synthesis and Characterization Data for PMMA Microspheres Prepared by Photoinitiated RAFT Dispersion Polymerization of MMA in Ethanol/Water Mixtures with PEGMA as the Stabilizer under Different Reaction Conditions entry

MMA, wt %a

PEGMA, wt %b

Darocur 1173, wt %b

DDMAT, wt %b

ethanol/water (w/w)

dn, μm

CVd, %c

1 2 3 4 5 6 7 8 9 10

10 10 10 10 10 10 10 10 10 10

2.5 5 2.5 2.5 2.5 1.25 5 10 2.5 2.5

3 3 3 3 3 3 3 3 3 3

0 0 0.25 0.5 0.75 0.5 0.5 0.5 0.5 0.5

40/60 40/60 40/60 40/60 40/60 40/60 40/60 40/60 35/65 45/55

− − 1.08 1.09 1.08 1.11 − − 1.13 1.02

− − 2.4 2.7 2.4 4.0 − − 3.2 8.4

11 12 13 14

10 5 15 15

2.5 2.5 2.5 2.5d

3 3 3 3

0.5 0.5 0.5 0.5

50/50 40/60 40/60 40/60

1.53 0.66 − 0.82

2.8 2.1 − 1.2

particle morphology polydisperse, smooth surface polydisperse, rough surface smooth surface smooth surface rough surface polydisperse, smooth surface rough surface network structure rough surface mixed smooth particles and rough particles rough surface smooth surface polydisperse, rough surface smooth surface

a

The amount of MMA was relative to the reaction mixture. bThe amounts of other reactants were relative to MMA. PEGMA2000, unless otherwise noted. cCVd is defined in eq 1. dPEGMA4000.

Yang et al.33 synthesized a series of polyacid macromonomers via a three-step route, first a range of well-defined poly(2-hydroxypropyl methacrylate) homopolymers were prepared by ATRP using a tertiary amine-functionalized initiator, and then quaternized the tertiary amine end group with 4-vinylbenzyl chloride, followed by esterification of the hydroxyl groups using excess succinic anhydride. They used these stabilizers for both emulsion and dispersion polymerization. In the dispersion polymerization of styrene in ethanol, particles with a narrow size distribution could be obtained under some reaction conditions. The particle sizes were small, with diameter ranging from 216 to 476 nm. In this paper, we report a photoinitiated RAFT dispersion polymerization using commercially available oligomeric macromonomer methoxy-poly(ethylene glycol) methacrylates (primarily, PEGMA2000, Mn = 2000 g/mol, EO45) as the hydrophilic steric stabilizer. We were able to identify a set of reaction conditions where PMMA microspheres were obtained with a very narrow size distribution. This was a one-pot, onestep reaction at room temperature, and allowed us to obtain carboxylated particles by incorporating small amounts of methacrylic acid (MAA) as a comonomer in the reaction. More surprisingly, we found that the carboxyl-functional microspheres prepared by this method were effective substrates for the covalent attachment of proteins, in spite of the PEG corona on the particle surface. This is not only an important step forward for the synthesis of particles useful for bead-based immunoassays, but this method provides a prototype for the synthesis of reactive polymer particles with functional macromonomers as the stabilizers.

10000 g/mol) as the stabilizer. They investigated the effect of monomer concentration, stabilizer concentration and stabilizer type on particle size and morphology. The size distributions of the particles they obtained were relatively broad. Chen et al.30 examined the dispersion polymerization of styrene using a poly(N-isopropylacrylamide) (PNIPAM) macromonomer in ethanol−water mixtures in the presence of silver nitrate. They obtained rather uniform polystyrene particles with diameters in the range of 480 to 1250 nm and PDI values of 1.01 to 1.02. Li et al.31 used a commercially available poly(ethylene oxide) macromonomer methoxy-poly(ethylene glycol) methacrylate (Mn = 2000 g/mol, EO45) as the reactive stabilizer in two-stage atom transfer radical polymerization (ATRP) dispersion polymerization of styrene, and only polydisperse particles were obtained under their reaction conditions. The Armes group has carried out extensive research on macromonomer synthesis and the application of these macromonomers in both emulsion polymerization and dispersion polymerization. Emulsion polymerization led to particles with submicrometer diameters, whereas they obtained microspheres by dispersion polymerization. Unlike the papers cited above that used electron microscopy to determine particle size, the Armes group employed disk centrifuge photosedimentometry (DCP) and dynamic light scattering (DLS) to measure particle sizes and size distributions. For example, Thompson et al.32 described a range of well-defined macromonomers obtained from glycerol monomethacrylate by ATRP. Submicrometer-sized and micrometer-sized polystyrene latexes were synthesized by emulsion and alcoholic dispersion polymerization, and micrometer-sized poly(2-hydroxypropyl methacrylate) latexes were obtained by aqueous dispersion polymerization. However, the particle size distributions were relatively broad compared to the use of conventional polymeric stabilizers. McKee et al.20 synthesized a PNIPAM polymer by RAFT polymerization and transformed part of this sample into a PNIPAM macromonomer. In the dispersion polymerization of styrene in methanol, using the PNIPAM RAFT polymer as the stabilizer, they obtained a bimodal particle size distribution, whereas the PNIPAM macromonomer gave particles with a monomodal but somewhat broad size distribution.

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EXPERIMENTAL SECTION

Materials. Absolute ethanol, 2-(dodecylthiocarbonothioylthio)-2methylpropionic acid (DDMAT, Aldrich), methyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate (Aldrich), poly(ethylene glycol) methyl ether methacrylate (PEGMA2000, 50 wt % solution, Mn = 2000 g/mol), (PEGMA500, Mn = 500 g/mol), (PEGMA4000, Mn = 4000 g/mol), all from Aldrich, 2-Hydroxy-2-methylpropiophenone (Darocur 1173, Aldrich), 4-Morpholineethanesulfonic acid (MES, Aldrich) were used without further purification. Methyl methacrylate (MMA, Aldrich) was passed through a column of basic alumina oxide 6858


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(Aldrich) prior to storage under refrigeration at 4 °C. Water was purified through a Milli-Q purification system. Bovine serum albumin (BSA), N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Goat IgG and Alexa Fluor 488 donkey anti-goat IgG (H+L) were purchased from Life Technologies. Instrumentation. A Hitachi S-5200 field emission scanning electron microscope was used at 1 kV operating voltage to measure particle size. The particles were diluted with distilled water, and a drop was placed on a mica film and allowed to dry. The SEM images were analyzed by the software program ImageJ (NIH, USA). For particles with a narrow size distribution, at least 100 particles were counted in several images. Confocal fluorescence microscopy images of the polymer particles were obtained with a Leica TCS SP2 confocal laser scanning microscope. Photoinitiated Dispersion Polymerization of MMA Using PEGMA as the Reactive Stabilizer. In a typical experiment, 10 wt % monomer (MMA, 2.0 g) relative to the system, 2.5 wt % macromonomer (PEGMA2000, 0.05 g) relative to MMA, 0.5 wt % RAFT agent (DDMAT, 0.01 g) relative to MMA and 3 wt % photoinitiator (Darocur 1173, 0.06 g) relative to MMA were dissolved in an ethanol/water mixture (40/60, w/w) under magnetic stirring to form a homogeneous solution. The mixture was gently purged with nitrogen for 15 min, and then sealed. A LED UV lamp (λ = 365 nm, and the light intensity 0.8 mW/cm2) was employed to irradiate the reaction mixture from the top of the reaction cell. The reaction mixture turned turbid after 90 s UV irradiation. After 3 h irradiation, a stable dispersion was formed without any observable coagulum. The obtained product was sedimented by centrifugation and washed three times with an ethanol/water (40/60, w/w) mixture. The dispersion was diluted with distilled water and a sample was prepared on a mica film for SEM analysis. The particles were not washed prior to preparing samples for SEM. A list of the reaction conditions employed in this study, and the particle sizes obtained are listed in Table 1. For experiments to monitor the time evolution of the particle size, the reaction conditions of entry 4 in Table 1 were employed. Aliquots (1 mL) were taken from the reaction with a microsyringe under nitrogen atmosphere at different time intervals. Each aliquot was weighed (W0); then the particles were separated by centrifugation and dried to a constant weight (W1). The particle yield (%) was calculated from the expression

particle yield (%) = (10W1/ W0) × 100

ethanol/water (40/60, w/w) mixture, and an aliquot was examined by SEM. In a typical two-stage dispersion polymerization, MMA (2.0 g), PEGMA2000 (0.05 g, 2.5 wt % relative to MMA), DDMAT (0.01 g, 0.5 wt % relative to MMA), and Darocur 1173 (0.06 g, 3 wt % relative to MMA) were dissolved in ethanol/water (7.2 g/10.8 g, 40/60, w/w). The mixture was purged with nitrogen and irradiated for 3 h as described above. Then a solution containing MMA (1.5 g), MAA (0.04 g), Darocur 1173 (0.06 g) and ethanol/water (3.2 g/4.8 g) was added to the reaction and irradiated for another 3 h. The product was sedimented, washed three times with the ethanol/water (40/60, w/w) mixture, and an aliquot was examined by SEM. Bioconjugation with Proteins. An aliquot of a carboxylfunctional PMMA particle dispersion (40 μL, solids content 9 wt %, prepared by the two-stage method as described above) was washed twice with 500 μL of MES buffer (pH 5.5, 100 mM) in a 2.0 mL Eppendorf tube with a slanted bottom, and the particles were redispersed in 400 μL of MES buffer. A solution of EDC and NHS in MES buffer (100 μL, containing 8 mg of EDC and 22 mg of NHS) was added to the vial with gentle vortexing for 30 min, then the particles were sedimented by centrifugation at 8000 × g and washed twice with 500 μL PBS buffer. The activated particles were redispersed in 1000 μL of PBS buffer, then split into two 1.5 mL centrifuge tubes, which contained either 40 μL of goat IgG solution (5 mg/mL in PBS) or 40 μL of BSA solution (5 mg/mL in PBS). Both samples were incubated with gentle vortexing for 3 h. The samples were then sedimented at 8000 × g and resuspended in PBS. This washing process was repeated twice more, and the supernatant was discarded. The washed particles were redispersed in 500 μL PBS (containing 0.5 wt % BSA), and stored at 4 °C overnight. Before the conjugation with anti-IgG, both samples were washed twice with 500 μL PBS to remove excess BSA, and redispersed in 480 μL PBS. Then a solution of Alexa Fluor 488 donkey anti-goat IgG (20 μL, 2 mg/mL in PBS) was added to both tubes, and incubated for 3 h. The samples were then sedimented at 8000 × g and resuspended in PBS. This washing process was repeated twice more, and the supernatant was discarded. The samples were finally redispersed in 500 μL PBS and stored at 4 °C. As a control experiment, we also carried out the bioconjugation protocol without EDC activation. An aliquot of a carboxyl-functional PMMA particle dispersion (20 μL, solids content 9 wt %, prepared by the two-stage method as described above) was washed twice with 500 μL PBS, and finally redispersed in 500 μL PBS. Then a goat IgG solution (5 mg/mL in PBS) was added to the dispersion, and the sample was incubated with gentle vortexing for 3 h. The particles were purified as described above, treated with a solution of Alexa Fluor 488 donkey anti-goat IgG (20 μL, 2 mg/mL in PBS), and washed again. The particles were finally redispersed in 500 μL PBS buffer and stored at 4 °C.

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In Situ Seeded Photoinitiated RAFT Dispersion Polymerization of MMA. In a typical experiment, 10 wt % monomer (MMA, 2.0 g) relative to the system, 2.5 wt % macromonomer (PEGMA2000, 0.05 g) relative to MMA, 0.5 wt % RAFT agent (DDMAT, 0.01 g) relative to MMA and 3 wt % photoinitiator (Darocur 1173, 0.06 g) relative to MMA were dissolved in an ethanol/water mixture (7.2 g/ 10.8 g, 40/60, w/w) to form a homogeneous solution. The mixture was purged with nitrogen and irradiated as described above. At the same time, a solution containing MMA (2.0 g), Darocur 1173 (0.06 g) and ethanol/water (3.2 g/4.8 g) was prepared. After 3 h of UV irradiation, the UV lamp was turned off, and the new MMA solution was added to the reaction mixture. Then the UV lamp was turned on, and the mixture was irradiated for another 3 h. The product was precipitated by centrifugation and washed three times with an ethanol/ water (40/60, w/w) mixture. The dispersion was diluted with distilled water and then dropped on a mica film for the SEM measurement. Preparation of Carboxyl-Functional PMMA Microspheres. In a typical one-stage dispersion polymerization, MMA (2.0 g), PEGMA2000 (0.05 g, 2.5 wt % relative to MMA), MAA, (0.04 g, 2 wt % relative to MMA), DDMAT (0.01 g, 0.5 wt % relative to MMA), and Darocur 1173, (0.06 g, 3 wt % relative to MMA) were dissolved in ethanol/water (40/60, w/w). The mixture was purged with nitrogen and irradiated as described above. The reaction mixture turned turbid after 90 s of UV irradiation. After 3 h of UV irradiation, a stable dispersion was formed without any observable coagulum. The product was sedimented by centrifugation and washed three times with the

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RESULTS AND DISCUSSION Photoinitiated dispersion polymerization is an attractive method to synthesize poly(methyl methacrylate) microspheres in a one-pot reaction. The reaction does not work very well unless a RAFT agent is added to the reactants (RAFT = reversible addition−fragmentation transfer). In the presence of the RAFT agent, the reaction occurs more slowly, but with much better control of particle size distribution. In recent work, we found that PMMA microspheres with a very narrow size distribution could be obtained by photoinitiated dispersion polymerization with PVP as the stabilizer, in which 2(dodecylthiocarbonothioylthio)-2-methylpropionic acid, a RAFT agent, was added at the beginning of the reaction.12,13 The need for a RAFT agent was explained in terms of the rapid decomposition of the photoinitiator under UV irradiation. This can lead to such rapid particle nucleation that they form in a colloidally unstable state, likely resulting in extensive coalescence of these primary particles. The RAFT agent 6859


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slows the reaction, and in this way, it is thought to allow sufficient graft copolymer formation to provide particle stability. In this way, the nucleation stage was controlled by the RAFT process. Here we examine the photoinitiated dispersion polymerization of MMA in ethanol−water in the presence of DDMAT as a RAFT agent, and with a methoxy PEG− methacrylate macromonomer (Mn = 2000 g/mol) as the stabilizer. For these dispersion polymerization reactions in the presence of a RAFT agent, we had to choose a RAFT agent which is photochemically stable at the irradiation wavelength. For example, Lu et al.34 reported that dithioesters decomposed rapidly under 365 nm UV irradiation, while trithiocarbonates were relatively stable. In this paper, we compare two trithiocarbonates as RAFT agents, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) and methyl 2(dodecylthiocarbonothioylthio)-2-methylpropionate, the corresponding methyl ester. As a photoinitiator, we used 2,2Hydroxy-2-methylpropiophenone, with the commercial name Darocur 1173. To set a baseline for comparison, we first examined the photoinitiated dispersion polymerization in the absence of the RAFT agent. Details are provided in Supporting Information. At low concentrations of PEGMA2000 as the macromonomer, we obtained only coagulum after 30 min UV irradiation. At somewhat higher PEGMA2000 concentration (2.5 wt % based on MMA), colloidally stable particles were obtained but the size distribution was broad (Figure S1a, Supporting Information). At higher PEGMA2000 concentration (5 wt %), the particles had a rough surface texture and a broad size distribution (Figure S1b). In Figure 1, we present SEM images and diameter histograms of PMMA microspheres prepared by photoinitiated dispersion polymerization of MMA (10 wt %) in ethanol−water (40/60 w/w) with 2.5 wt % PEGMA2000 (based on monomer) in the presence of DDMAT. In the different examples, the concentration of DDMAT was varied, but other reaction conditions were held constant. PMMA microspheres formed that were uniform in size. For example, with 0.25 wt % DDMAT (entry 3, Table 1), we obtained particles characterized by dn = 1.1 μm, CVd = 2.4%. An increase of DDMAT concentration to 0.5 and 0.75 wt % had almost no influence on the particle size and particle size distribution. For the two reactions with the lower amounts of DDMAT (0.25 wt %; and 0.5 wt %), the particle surfaces were smooth (Figure 1a,b). At the highest DDMAT concentration (0.75 wt %, entry 5, Table 1), however, the particle surfaces exhibited a noticeable roughness in SEM images (e.g., Figure 1c). It is gratifying that we were able to achieve particles with such a narrow size distribution. Curiously, when we carried out the corresponding photoinitiated RAFT dispersion polymerization with methyl 2(dodecylthiocarbonothioylthio)-2-methylpropionate as the RAFT agent, the particles obtained were not very uniform in their size distribution. The results shown in Figure S2 indicate that these particles have a noticeably narrower size distribution than those synthesized in the absence of a RAFT agent (Figure S1). The small difference in structure between DDMAT and its methyl ester had profound consequences on the course of the reaction. While we were fortunate to find conditions with DDMAT that led to very uniform microspheres, the main lesson from these experiments is how sensitive the reaction is to small changes in reactants. In the paragraphs that follow, we

Figure 1. SEM images and histograms of PMMA microspheres prepared by photoinitiated RAFT dispersion polymerization of MMA in an ethanol/water mixture (40/60, w/w) with 2.5 wt % PEGMA2000 as the stabilizer in the presence of different amounts of DDMAT: (a) 0.25 wt %, dn = 1.08 μm, CVd = 2.4%; (b) 0.5 wt %, dn = 1.09 μm, CVd = 2.7%; (c) 0.75 wt %, dn = 1.08 μm, CVd = 2.4%;. The concentration of photoinitiator was 3 wt % (relative to MMA) and the concentration of MMA was 10 wt % (relative to the reaction mixture). Scale bar: 2 μm.

explore other aspects of the photoinitiated RAFT dispersion polymerization in the presence of DDMAT. Variation of Reaction Conditions. Macromonomer Concentration and Chain Length. In the next set of experiments, we tested the influence of PEGMA2000 concentrations on particle formation in the presence of 0.5 wt % DDMAT. When the amount of PEGMA2000 in the reaction was 1.25 wt % (relative to MMA), PMMA particles with smooth surfaces were obtained, but the particles size distribution was relatively broad (Figure 2a, and Table 1, entry 6). The reaction maintained colloidal stability over the entire reaction. When the amount of PEGMA2000 was increased to 2.5 wt %, monodisperse PMMA particles with smooth surfaces were obtained (CVd = 2.7%; Figure 1b, entry 4, Table 1). At 5 wt % (Figure 2b) and 7.5 wt % (Figure 2c) the particle surfaces became rough and the particles had irregular shapes. By eye, the size distributions in the SEM images appeared to be narrow. However, when the PEGMA2000 concentration was increased to 10 wt %, the particles became polydisperse (e.g., Figure 2d). At higher PEGMA2000 concentrations (12.5 or 15 wt %), the reaction no longer yielded spherical particles. Rather, we found network structures (Figure 2e,f) that appeared to have been formed by aggregation of small particles. In terms of our objectives, it appears that in the photoinitiated RAFT dispersion polymerization of MMA with PEGMA2000 as the stabilizer, 2.5 wt % PEGMA2000 is an appropriate concentration for obtaining monodisperse polymer particles. This amount can be contrasted to the much higher amount of PVP as stabilizer (commonly 15 wt % based on monomer) needed for dispersion polymerization of styrene or MMA. In that case, only a small fraction of the PVP becomes 6860


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These results indicate that the molecular weight of the macromonomer is also an important parameter for obtaining PMMA microspheres with a narrow size distribution. The molecular weight of the macromonomer should be high enough to provide colloidal stability throughout the reaction. If the molecular weight of macromonomer is too low, it cannot provide sufficient steric stabilization to the growing particles, and this leads to coagulation. Solvent Composition. For the experiments described above, reactions were carried out in a 40/60 (w/w) ethanol−water mixture. Here we examine the influence of solvent composition on the reaction. As a reference, we note that in the 40/60 solvent mixture, with 10 wt % MMA in the presence of 2.5 wt % PEGMA2000 and 0.05 wt % DDMAT (both based on monomer) uniform particles with dn = 1.1 μm, CVd = 2.7% were obtained. Figure 3 shows the SEM images and diameter histograms of PMMA microspheres prepared with different ethanol/water ratios (w/w). In a slightly more polar medium, ethanol/water =35/65 (entry 9, Table 1), the PMMA particles were similar in size, with a somewhat broader size distribution (dn = 1.1 μm; CVd of 3.2%), and with a noticeable surface roughness in the SEM images (e.g., Figure 3a). In a slightly less polar medium, ethanol/water =45/55 (entry 10, Table 1), the product was a mixture of smooth particles and rough particles, and the particle diameter also exhibited a bimodal distribution (Figure 3b). When the ethanol/water ratio (w/w) further increased to 50/50 (entry 11, Table 1), all the particles became rough, and the mean particle diameter increased to 1.5 μm, but the size distribution remained narrow (CVd = 2.8%, Figure 3c). Monomer Concentration. In this section, we examine the consequences of varying MMA concentration from 5 to 15 wt %, with other reaction conditions being held constant. Figure 3d shows an SEM image and the diameter histogram of PMMA microspheres prepared with 5 wt % MMA (entry 12, Table 1). Uniform but smaller particles were obtained (dn= 0.66 μm, CVd = 2.1%) characterized by a smooth particle surface, compared to those obtained at 10 wt % PMMA (dn= 1.1 μm, CVd = 2.7%) described above. When the concentration of MMA was increased to 15 wt % (Figure 3e, entry 13, Table 1), only polydisperse irregular particles were obtained. These particles had rough surfaces. The main conclusion from these experiments is that the reaction is very sensitive to the amount of macromonomer present, is robust to small changes in solvent composition, and is able to tolerate some variation in the amount of monomer in the reaction. The sensitivity to macromonomer content is likely related to the delicate role it plays in particle nucleation. The effect of solvent composition and monomer content is somewhat easier to explain. More ethanol or more MMA in the reaction increases the solvency of the medium for both PMMA and for its copolymer with the macromonomer. There are many examples of dispersion polymerization where increasing the solvency of the medium for the polymer leads to an increase in particle size. This phenomenon is attributed to a longer chain length of polymer in solution preceding its collapse, and this in turn leads to fewer nuclei in the reaction. Solvent swelling of the particles can lead to irregular shrinkage upon drying. This may be the source of particle roughness seen under some reaction conditions. When the monomer concentration is too high (e.g., 15 wt % here), nucleation becomes difficult because of the high solubility of PMMA chains in the reaction medium, resulting in a broad particle size distribution.

Figure 2. SEM images and histograms of PMMA microspheres prepared by photoinitiated RAFT dispersion polymerization of MMA in an ethanol/water mixture (40/60, w/w) in the presence 0.5 wt % DDMAT with different amounts of PEGMA2000: (a) 1.25 wt %; (b) 5 wt %; (c) 7.5 wt %; (d) 10 wt %; (e) 12.5 wt %; (f) 15 wt %; and (g) 2.5 wt % PEGMA4000. An SEM image of the particles formed in the presence of 2.5 wt % PEGMA2000 is presented in Figure 1b. In all reactions, the concentration of photoinitiator was 3 wt % (relative to MMA) and the concentration of MMA was 10 wt % (relative to the reaction mixture). Scale bars: 2 μm.

grafted to the particles, and the unreacted polymer has to be removed at the end of the reaction. The success of these reactions prompted us to examine the effect of PEG chain length on the reaction. Thus, we carried out photoinitiated RAFT dispersion polymerizations of MMA with a shorter (PEGMA500) and a longer (PEGMA4000) macromonomer as the stabilizer. For PEGMA500, a reaction was attempted with 2.5 wt % macromonomer, and with other reaction conditions held the same as those described above. After 1.5 h of UV irradiation, a coagulum formed and the reaction was terminated. In an attempt to overcome this problem, we increased the amount of PEGMA500 to 15 wt %. Again, we had no success in obtaining colloidally stable particles as a product. In contrast, the longer macromonomer (PEGMA4000) worked well. We carried out the photoinitiated RAFT dispersion polymerization in the presence of 2.5 wt % PEGMA4000, and the reaction product remained colloidally stable during the whole process. As shown in Figure 2g, PMMA microspheres with a very narrow size distribution were obtained (dn = 0.82 μm, CVd = 1.2%). 6861


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Figure 4. SEM images of PMMA microspheres prepared by photoinitiated RAFT dispersion polymerization of MMA in ethanol/ water mixture (40/60, w/w) at different UV initiation times (marked in the images). The concentration of PEGMA2000 was 2.5 wt % (relative to MMA), the concentration of DDMAT was 0.5 wt % (relative to MMA), the concentration of photoinitiator was 3 wt % (relative to MMA), and the concentration of MMA was 10 wt % (relative to the reaction mixture). Scale bar: 2 μm.

Figure 3. SEM images and histograms of PMMA microspheres prepared by photoinitiated RAFT dispersion polymerization of MMA with different ethanol/water ratios (w/w): (a) 35/65, dn = 1.13 μm, CVd = 3.2%; (b) 45/55, dn = 1.02 μm, CVd = 8.4%; (c) 50/50, dn = 1.53 μm, CVd = 2.8%. An SEM image of the particles formed by photoinitiated RAFT dispersion polymerization with 40/60 ethanol/ water ratio (w/w) is presented in Figure 1b.; (d, e) SEM images and histograms of PMMA microspheres prepared by photoinitiated RAFT dispersion polymerization of MMA in an ethanol/water mixture (40/ 60, w/w) with different initial monomer concentrations: (d) 5 wt %, dn = 0.66 μm, CVd = 2.1%; (e) 15 wt %. For the image of 10 wt % refer to Figure 1b. Scale bar: 2 μm.

Evolution of Particle Size During the Reaction. To further investigate the formation process of the polymer particles prepared by photoinitiated RAFT dispersion polymerization with PEGMA2000 as the stabilizer, we followed the evolution of particle size by SEM. Samples were withdrawn at different irradiation times, diluted with an ethanol/water (40/ 60, w/w) mixture to stop the reaction, and then examined by SEM. We focus on conditions found to be optimal for the reaction through the studies described above: 10 wt % MMA, 2.5 wt % PEGMA2000, and 0.5 wt % DDMAT (both relative to monomer), 40/60 ethanol/water (w/w). Figure 4 shows SEM images of PMMA microspheres obtained at different irradiation times, and Figure 5 (top) shows plots of product yield and particle diameter versus UV irradiation time. At 5 min, the

Figure 5. (Top) Plots of particle diameter and product yield versus UV irradiation time for PMMA microspheres prepared by photoinitiated RAFT dispersion polymerization of MMA. (Bottom) Particle yield versus particle volume for photoinitiated RAFT dispersion polymerization of MMA. The x-axis indicates the particle yield measured by gravimetry, and the y-axis represents the particle volume calculated from the diameter of the particles as measured by SEM. The concentration of PEGMA2000 was 2.5 wt % (relative to MMA), the concentration of DDMAT was 0.5 wt % (relative to MMA), the concentration of photoinitiator was 3 wt % (relative to MMA), and the concentration of MMA was 10 wt % (relative to the reaction mixture).

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SEM images and diameter histograms of the seeds obtained after the initial 3 h irradiation, as well as the final product. PMMA microspheres with smooth surfaces and a narrow size distribution were obtained in both cases. The mean particle diameter increased from 1.1 μm (CVd = 2.7%) after the first stage to 1.4 μm (CVd = 3.6%) after the second stage. The increase of the particle volume (107%) is equal to the additional amount of monomer (100%) added in the second stage. This result suggests that one can control particle size by adding different amounts of monomer in seeded photoinitiated RAFT dispersion polymerization. Carboxyl-Functional PMMA Microspheres. Carboxylic acid groups are important surface-functional groups for particles intended for biomedical or biodiagnostic applications, because they can be used for the attachment of proteins or other biomolecules. These carboxyl groups are normally introduced by adding a carboxylate functional comonomer such as acrylic acid (AA) or methacrylic acid (MAA) into the particle synthesis reaction.7,12,35,36 In our previous research on photoinitiated RAFT dispersion polymerization of MMA with PVP as the stabilizer, the presence of functional comonomers (e.g., 2-hydroxypropyl methacrylate, methacrylic acid, acrylic acid, and glycol methacrylate) had no influence on the course of the reaction or the particle size distribution.12 Here we encountered a different result with PEGMA2000 as the stabilizer. When we carried out a one-stage photoinitiated RAFT dispersion polymerization with PEGMA2000 in the presence of 2 wt % MAA based on MMA, we could only obtain polydisperse particles as shown in Figure 7a. This result suggests that the presence of MMA at the beginning of the reaction disturbed the nucleation stage of the dispersion polymerization. It is possible that incorporation of MAA groups into the P(PEGMA-co-MMA) stabilizer increases its hydro-

particle yield was low (1.6%). Spherical particles had formed with a diameter dn = 0.37 μm, but with a relatively broad size distribution (CVd = 7.0%). As the reaction time increased to 60 min, the particle yield reached 34%. At this point, spherical particles with a diameter of 0.80 μm were obtained, and the particles became much more monodisperse (CVd = 2.8%). When the reaction time further increased to 180 min, the particle yield reached 86%, the particle diameter increased to 1.02 μm, and the size distribution remained narrow (CVd = 2.6%). In Figure 5 (bottom), we plot particle yield against particle volume and find that particle yield tracks linearly with particle volume. If one assumes that the particles are composed only of PMMA and that all the MMA reacted ended up in the particles, then the linearity of this plot indicates that the number of particles in the reaction remained constant. No new particles were formed as the reaction proceeded. In Situ Seeded Photoinitiated RAFT Dispersion Polymerization. In many types of dispersion polymerizations, it is problematic to introduce functional monomers at the beginning of the reaction because they interfere with the nucleation step. This problem disappears if one waits until nucleation is complete and the number of particles in the reaction becomes constant before adding the functional monomer. We have referred to this delay in the addition of problematic monomers as “two-stage” dispersion polymerization and note that it is a kind of seeded polymerization process. This type of seeded polymerization is also useful for increasing the size of polymer particles by adding more monomer to a reaction containing a fixed number of growing particles. In this section, we examine a two-stage polymerization in which additional MMA is added to the reaction after 3 h photoirradiation. The initial reaction mixture corresponds to entry 4 in Table 1 (MMA, 2.0 g, PEGMA2000, 0.05 g, DDMAT, 0.01 g, Darocur 1173, 0.06 g dissolved in ethanol/ water (7.2 g/10.8 g, 40/60, w/w)). At this point, a degassed solution containing MMA, (2.0 g), photoinitiator (0.06 g), ethanol (3.2 g) and water (4.8 g) was added, and then the solution was irradiated for an additional 3 h. Figure 6 shows the

Figure 7. (a) SEM image of carboxyl functional PMMA particles prepared by photoinitiated RAFT dispersion polymerization with 0.04 g MAA added at the beginning: dn = 1.07 μm; CVd = 34%. (b) SEM image of PMMA seeds prepared by photoinitiated RAFT dispersion polymerization with typical condition described in the Experimental Section (entry 4, Table 1): dn = 1.05 μm; CVd = 2.4%. (c) SEM image of carboxyl functional PMMA microspheres prepared by in situ seed photoinitiated RAFT dispersion polymerization with 0.04 g MAA added in the second stage: dn = 1.37 μm; CVd = 2.3%. Scale bar: 2 μm.

Figure 6. (a) SEM image and histogram of PMMA seeds prepared by photoinitiated RAFT dispersion polymerization with typical condition described in the Experimental Section (entry 4, Table 1): dn = 1.09 μm CVd = 2.7%. (b) SEM image and histogram of PMMA microspheres prepared by in situ seed photoinitiated RAFT dispersion polymerization with another portion of monomer (2.0 g) added to the dispersion: dn = 1.39 μm; CVd = 3.6%. Scale bar: 2 μm. 6863


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philicity and interferes with its ability to stabilize PMMA particles at the beginning of the reaction. In an attempt to overcome this problem, we carried out a two-stage (seeded) photoinitiated RAFT dispersion polymerization with MAA added in the second stage. The PMMA seeds were prepared as described in entry 4, Table 1. After 3 h of UV irradiation, we added a degassed solution containing MMA (1.5 g), MAA (0.04 g), photoinitiator (0.06 g), ethanol (5.4 g), and water (8.1 g). The reaction mixture was then irradiated for another 3 h. The product was sedimented and then washed three times with the ethanol/water (40/60, w/w) mixture. Figure 7b shows the SEM image of the PMMA seed microspheres prepared in the first stage of the reaction, characterized by dn = 1.05 μm, (CVd = 2.4%). After the second stage reaction, the particle diameter increased to 1.37 μm (CVd = 2.3%, Figure 7c). Thus, the problems associated with the introduction of MAA into the one-stage dispersion polymerization with PEGMA2000 as the stabilizer can be overcome with a two-stage process. The mean number of carboxyl groups per particles was determined by simultaneous potentiometric and conductometric titration. The titration curve is shown in Figure S3. On the basis of the titration result, we calculate an average of 7.9 × 106 carboxyl groups per particle. Attachment of Proteins to the Surface of the Particles. To be able to use this type of polymer microsphere in immunoassays, we need to be able to attach antibodies covalently to the particle surface. While the carboxylated particles described above contain a substantial number of titration-accessible −COOH groups, we need to test whether the PEG chains with Mn = 2000 g/mol grafted at the particle surface interfere with covalent attachment of proteins. To proceed, two samples of EDC-activated PMMA microspheres were treated in parallel either with goat IgG (40 μL, 5 mg/mL) or with bovine serum albumin (BSA) (40 μL, 5 mg/mL) in PBS buffer. The EDC-activated particles treated with BSA serve as our negative control. Each sample was allowed to react under vortexing for 3 h at room temperature and then washed three times with PBS buffer. After blocking the microspheres with 0.5 wt % BSA/PBS buffer for 24 h, the two particle samples were incubated 3 h with Alexa Fluor 488 donkey anti-goat IgG (20 μL, 2 mg/mL). After several washes with PBS buffer, the samples were analyzed by confocal fluorescence microscopy (CFM). Figure 8b shows the immunofluorescence image in the green channel (488 nm excitation) of the antibody-labeled microspheres subsequently treated with the dye-labeled donkey anti-goat IgG. The corresponding negative control experiment, with the BSA-labeled microspheres treated with dye-labeled donkey anti-goat IgG is shown in Figure 8d. These images show that there is a bright fluorescent signal from the IgGlabeled microspheres in Figure 8b, whereas there is nearly no detectable fluorescent signal on the negative-control microspheres in Figure 8d. In order to estimate the mean number of anti-goat IgG molecules attached per microbead, we carried out an experiment of dye-labeled antibody. After each reaction, the particles were sedimented and the supernatant was analyzed by UV−vis spectroscopy for the concentration of unbound Alexa Fluor 488 donkey anti-goat IgG remaining in solution. The absorbance at 494 nm was compared to the standard curve in Figure S5. Details are provided in the Supporting Information. In this way, we determined that there were on average 1.1× 104

Figure 8. Optical (a) and CFM (b) (488 nm excitation) images of goat IgG-coated PMMA particles, treated with Alexa Fluor 488 donkey anti-goat IgG; optical (c) and CFM (d) (488 nm excitation) images of BSA-coated PMMA particles, treated with Alexa Fluor488 donkey anti-goat IgG; optical (e) and CFM (f) (488 nm excitation) images of PMMA particles, which was mixed directly with goat IgG, and then treated with Alexa Fluor 488 donkey anti-goat IgG. Scale bar: 4 μm.

donkey anti-goat antibodies bound per goat IgG-coated PMMA microsphere (Table S1). We also carried out a control experiment to ensure that the proteins were covalently attached to the particle surface. A sample of carboxylated PMMA microspheres particles was mixed directly with goat IgG (40 μL, 5 mg/mL) without EDC activation, and vortexed for 3 h as described above. The particles were then washed three times with PBS buffer, blocked with 0.5 wt % BSA in PBS buffer for 24 h, and finally incubated 3 h with Alexa Fluor 488 donkey anti-goat IgG (20 μL, 2 mg/mL). After several washes with PBS buffer, the samples were analyzed by CFM. Figure 8f shows the immunofluorescence image in the green channel (488 nm excitation), and only a weak fluorescent signal from the particles was observed. Thus, we can conclude that the carboxyl groups on the particle surface are accessible for bioconjugation in spite of the presence of the PEG corona on the particles. These results demonstrate that the particles prepared by photoinitiated RAFT dispersion polymerization with PEGMA2000 as the stabilizer can serve as a support for bead-based bioassays. 6864


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SUMMARY In this paper, we report a photoinitiated dispersion polymerization of MMA with a macromonomer (methoxy-poly(ethylene glycol) methacrylate, Mn = 2000 g/mol, EO45) as the stabilizer. The presence of a carboxyl trithiocarbonate RAFT agent (DDMAT) was crucial for obtaining PMMA microspheres with a narrow size distribution, and the detailed structure of the RAFT agent had a big effect on the course of the reaction. Within the range of concentrations of DDMAT that we examined (0.25 to 0.75 wt %), PMMA microspheres with narrow size distributions were obtained with similar mean diameters. We also studied the effect of other reaction conditions (e.g., macromonomer concentration, solvent composition and monomer concentration) on particle morphologies. We found that the reaction was very sensitive to the amount of macromonomer, robust to small changes in solvent composition, and able to tolerate some variation in the amount of monomer in the reaction. To further investigate the formation process of the polymer particles prepared by photoinitiated RAFT dispersion polymerization with PEGMA as the stabilizer, we followed the evolution of particle size by SEM. We found that particle yield tracked linearly with particle volume, indicating that no new particles formed during the reaction. We also developed a two-stage (seeded) photoinitiated RAFT dispersion polymerization protocol. This approach allowed us to vary particle size, and, with methacrylic acid added in the second stage, to prepare carboxylated PMMA microspheres. To test the utility of these carboxylated microbeads in immunoassays, we used EDC activation to attach goat IgG to the particles. Its presence on the particles was detected by confocal fluorescence measurements with Alexa Fluor 488 donkey anti-goat IgG as the reporter. In order to estimate the amount of goat IgG that could be attached to the particle surface, we repeated the secondary antibody experiment with the dye-labeled donkey anti-goat IgG, monitoring its depletion from the supernatant by UV−vis spectroscopy. In this way we found that that on average 1.1 × 104 donkey anti-goat IgG molecules were bound per goat IgG-coated PMMA microsphere. These results are different than those reported previously for carboxylated PS microspheres with a poly(Nvinylpyrrolidone) corona on the surface, where we had only limited success in covalently attaching antibodies to the particles. The carboxyl-functional microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization can be used as effective substrates for the covalent attachment of proteins, in spite of the PEG corona on the particle surface.

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ACKNOWLEDGMENTS The authors thank NSERC Canada and DVS Sciences for their financial support. JT thanks the Chinese Scholarship Council for a scholarship to come to the University of Toronto. J.T. and Z.Z. thank the National Natural Science Foundation of China (Grants 20974126 and 21174165).

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ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details for synthesis and characterization of the PMMA microspheres, pH and conductometric titration of carboxylated microspheres, quantification of antibodies per microbead by UV−vis spectrometry, and derivation of the relationship between CVd and PDI. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(M.A.W.) E-mail: [email protected]. Notes

The authors declare no competing financial interest. 6865


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